Superiority of systemic bleomycin to intradermal HOCl for the study of interstitial lung disease

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, immune dysregulation, and multi-organ fibrosis. Interstitial lung disease (ILD) is a complication of SSc and a leading cause of SSc-death. The administration of hypochlorous acid (HOCl) intradermally in the mouse (HOCl-SSc) purportedly shows several features typical of SSc. We studied the model by injecting BALB/c mice daily intradermally with HOCl for 6-weeks, an exposure reported to induce lung fibrosis. On day 42, the skinfold thickness and the dermal thickness were two and three times larger respectively in the HOCl group compared to controls. HOCl treatment did not result in histological features of pulmonary fibrosis nor significant changes in lung compliance. Automated image analysis of HOCl mice lungs stained with picrosirius red did not show increased collagen deposition. HOCl injections did not increase pulmonary mRNA expression of pro-fibrotic genes nor induced the production of serum advanced oxidation protein products and anti-topoisomerase 1 antibodies. Immune cells in bronchoalveolar lavage fluid (BALF) and whole lung digests were not increased in HOCl-treated animals. Since lung fibrosis is proposed to be triggered by oxidative stress, we injected HOCl to Nrf2−/− mice, a mouse deficient in many antioxidant proteins. Lung compliance, histology, and BALF leukocyte numbers were comparable between Nrf2−/− mice and wild-type controls. We conclude that the HOCl-SSc model does not manifest SSc-lung disease.


Animals
The ' Animal Research: Reporting of In Vivo Experiments' (ARRIVE) guidelines were used 20 .For the HOCl-SSc mouse model, female BALB/c mice (6-8-week-old) were purchased from Charles River Laboratories (St.Constant, QC, Canada), while Nrf2 −/− mice on a C57BL/6 background and corresponding wild type mice were purchased from Jackson Laboratories (Bar Harbor, Maine, United States).For the BLM mouse model, male C57Bl/6, 9-10-week-old mice were purchased from Jackson Laboratories.Animals were housed in a conventional animal facility at the Research Institute of the McGill University Health Centre (RI-MUHC).Animals were treated in accordance with the guidelines of the Canadian Council of Animal Care (CCAC) and protocols were approved by the Animal Care Committee of McGill University.

HOCl-SSc model: experimental design and preparation of hypochlorous acid (HOCl) solution
To induce lung fibrosis, we followed reported methods 4,[9][10][11][12]15 . Mic were anesthetized with isoflurane (4% induction, 1.5-2.5% maintenance) and received intradermal injections of HOCl in two sites of the lower back (150 µL in each site, a total of 300 µL) five days a week for 6 weeks.After 6 weeks, injections were discontinued, and experimental readouts were assessed 9,11 .Additional experiments were conducted to test the effects of increasing the total volume of the injected solution to 400 µL (200 µL per site) and prolonging the duration of injections for 2 additional weeks (8 weeks total).Different brands of commercially available bleach were also tested.
Potassium dihydrogen phosphate (KH 2 PO 4 ) buffer solution was prepared at a concentration of 100 mM with a pH of 6.2, as previously established 11 .The KH 2 PO 4 solution was stored at 4 °C for a maximum of 3 months.Commercial bleach at a concentration of 4% was used.The bleach bottle was replaced every 3 weeks, once opened.The HOCl solution was prepared at a final concentration of 0.096% by addition of the buffer solution.The optical density was measured at a wavelength of 292 nm by spectrophotometry and was between 0.7 and 0.9 (arbitrary units).The HOCl solution was prepared fresh daily.The control group received injections of an equal volume of PBS 9,11 .

Skin thickness
Skin thickness was measured with a digital caliper (Mitutoyo 547-500S) at three different locations on the lower back of the mouse near the site of injections.The average of the 3 measurements was recorded.

Measurement of respiratory mechanics
To evaluate lung function, we followed methods previously described 21,22 .Mice were sedated with xylazine (8 mg/kg; intraperitoneally) and anesthetized with pentobarbital sodium (30 mg/kg; intraperitoneally).They were tracheostomized and an 18-gauge metal cannula was inserted and connected to a computer-controlled small animal ventilator (flexiVent™, SCIREQ, Montreal, PQ, Canada).Mice were mechanically ventilated at 150 breaths/minute with a positive end expiratory pressure (PEEP) of 3cmH 2 O. Next, a muscle relaxant was administered (rocuronium bromide; 2 mg/kg) intraperitoneally.This was followed by 3 min of mechanical ventilation

Quantification of dermal thickness
Masson's Trichrome or PSR stained slides of mouse skin sections were scanned using the Aperio Scanner and analyzed using the Aperio ImageScope software at 20 × magnification.Dermal thickness was measured from the dermal-epidermal junction to the dermal-adipose junction.Either two or three skin biopsies per mouse were used for analysis.Three random measurements were taken per section.

Advanced oxidation protein products (AOPPs)
The protocol used for obtaining serum was adapted from "Blood Collection and Sample Preparation for Rodents" (IDEX BioAnalytics) 27 .Blood from PBS and HOCl-injected mice was obtained after the completion of FlexiVent measurement by cardiac puncture and collected in tubes (BD Microtainer®).Tubes were centrifuged at 3800 rpm for 10 min (Eppendorf® centrifuge 5424).The serum was aliquoted in 1.5 mL Eppendorf tubes and stored at − 20 °C until the levels of AOPPs and anti-Scl-70 antibodies were determined.
Serum from PBS and HOCl-injected mice was diluted 1/20 in PBS and 200 μL was pipetted in a 96-well plate.A solution of chloramine T (100 μM) was used to prepare the standard curve.All samples and standards were run in duplicates.An aliquot of 10 μL of 1.16 M potassium iodide was added to each well, mixed and incubated for 5 min at room temperature.Next, 20 μL of acetic acid solution was added to each well to stop the reaction.Optical density was read at 340 nm on a microplate spectrophotometer (LKB Pharmacia 4050 UltroSpec II UV-Vis).AOPP content was calculated using the chloramine T standard curve 9 .

Anti-topoisomerase I autoantibodies (anti-Scl-70)
A mouse anti-Scl-70 ELISA kit (Signosis, EA-5205) was used.The ELISA was conducted following the specifications of the manufacturer.The kit contained a positive and negative control.Optical density was read at 540 nm on a microplate spectrophotometer.

Lung hydroxyproline assay
Lung hydroxyproline content was analyzed with a hydroxyproline colorimetric assay kit (Abcam, Ab222941) following manufacturer's instruction.

RNA isolation
The mouse right lung and an amount of skin of approximately 30 mg were dissected, transferred to 2 mL screwtop tubes (Heathrow Scientific HEA10060), snap frozen and stored at − 80 °C until the RNA isolation.Homogenization beads and lysis buffer with tris(2-carboxyethyl) phosphine (TCEP) was added to the frozen tissues and homogenized (607, Mini-BeadBeater-16). Lung and skin RNA were extracted using the Nucleospin® RNA kit (Macherey-Nagel™ 740955.50)and RNeasy Mini Kit (Qiagen, Inc), respectively, according to the manufacturer's instructions.The purity of RNA was verified by a microplate reader (Take3™ Micro-Volume Plate) using the Gen 5™ software.

Reverse transcription and real-time quantitative PCR
Following RNA isolation, cDNA was synthesized with the LunaScript® RT SuperMix Kit (NEB #E3010) according to the manufacturer's instructions using a thermocycler (Applied Biosystems™ Veriti™
Cells were fixed using the fixation reagent and diluent (Invitrogen™).All samples were acquired using the LSRFortessa x-20 flow cytometer.Analysis was performed using FlowJo V10 software.Fluorescence minus one (FMO) controls were used to set the upper boundary for background signal on the omitted label, and to identify and gate positive populations.The gating strategy is outlined in Supplementary Fig. 1.

Statistical analysis
GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis.Data are expressed as mean ± standard error of the mean (SEM).Non-parametric Mann-Whitney U test was used to compare two groups.Two-way ANOVA was used for analysis of P-V loops.A p-value less that 0.05 was considered statistically significant.

HOCl injections induce local skin fibrosis whereas BLM-MPs induce distal skin fibrosis
To assess skin fibrosis in HOCl-injected mice, we measured skinfold thickness with calipers, performed histological assessment of H&E and MT-stained sections, and evaluated mRNA expression of profibrotic genes.Compared to PBS-injected mice, HOCl mice had increased skin thickness at all timepoints (Supplementary Fig. 1a).H&Estained skin sections of HOCl-injected mice showed areas of epidermal hyperplasia, hyperkeratosis, and loss of adipose tissue and appendages.Those affected areas alternated with less or unaffected areas (Supplementary Fig. 1b).Inflammation extending into the superficial layer of the muscle bundles was also observed (Supplementary Fig. 1b).MT sections of HOCl-injected mice showed both areas of irregular and homogeneous collagen bundles in the dermis.Evidence of fibrosis was also observed in the deep reticular dermis (Fig. 1a).Histological assessment showed both HOCl and BLM-MP mice to have increased dermal thickness compared to PBS mice (Fig. 1b,g).However, HOCl skin thickness was only increased at the site of injections whereas BLM-MP mice had increased dermal thickness at a distance from the site of minipump implantation.Lastly, mRNA expression of Col1a1, Col3a1, and ACTA2 was elevated in HOCl treated mice (Fig. 1c-e).

BLM-MPs generates abnormalities in pulmonary histology and impairs lung function
The degree of lung injury induced by bleomycin was evaluated by histology, pulmonary function tests and micro-CT imaging.Four weeks post-pump implantation, BLM-MP mice had decreased lung compliance, increased resistance, and dynamic elastance compared to control animals (Fig. 3a-d).H&E-stained lung sections showed a predominantly inflammatory process with minimal evidence of fibrosis.The distribution of parenchymal injury was largely peripheral which resembles the pattern in interstitial lung disease (Fig. 3e).In the histology quantification, BLM-MP animals tend to have higher number of positive picrosirius red pixels and affected lung area (Fig. 3f-g).The presence of increased areas of attenuation on micro-CT corroborated the BLM-induced lung injury (Supplementary Fig. 3).

Inflammatory infiltrates in BALF and collagenase digests of lung tissue are similar in PBS and HOCl-injected mice
Inflammatory cells are reported to precede and mediate the development of fibrosis by releasing a variety of proinflammatory factors that inflict tissue injury [29][30][31][32] .Therefore, we tested whether intradermal HOCl injections induced lung inflammation.Evaluation of BALF cells showed no difference in total white blood cell counts and differential cell counts between PBS and HOCl-injected animals (Fig. 4a).Lung tissue digests were analyzed by flow cytometry in a separate group of mice.Representative contour plots of the flow cytometry gating strategy are shown in Supplementary Fig.

HOCl injections do not induce AOPPs or anti-Scl-70 antibodies production
HOCl purportedly leads to lung fibrosis by inducing oxidative stress in the skin that leads to AOPPs that propagate the injury to the lungs 9 .However, we did not observe increased concentrations of AOPPs in the sera of HOCl mice (PBS vs HOCl: 47.21 ± 11.62 vs 40.12 ± 10.87 µmol/L) (Fig. 5a).The development of fibrosis was also reported to be associated with the production of anti-Scl-70 antibodies 9 .HOCl mice were seronegative for anti-Scl-70 (PBS vs HOCl, arbitrary units: 0.19 ± 0.02 vs 0.21 ± 0.01) (Fig. 5b).These data fail to demonstrate systemic involvement following HOCl injections.

HOCl injections did not induce lung inflammation in Nrf2 −/− mice
It was previously shown that the Nrf2 pathway is involved in attenuating lung inflammation in bleomycininduced injury 33,34 .Therefore, in the absence of Nrf2, inflammatory responses are expected to be increased.We assessed whether Nrf2 −/− mice had increased airway inflammation by analyzing total and differential cell counts in BALF.Compared to wild type C57BL/6 HOCl mice, total cells were not increased in Nrf2 −/− HOCl mice (C57 HOCl vs Nrf2 −/− , absolute number of cells: 34,167 ± 2626 vs 30,400 ± 2064) (Fig. 7d).Moreover, there were no differences in the number of macrophages, eosinophils, neutrophils, and lymphocytes between these two groups.These data show that HOCl-generated oxidative stress does not result in the development of pulmonary inflammation following intradermal HOCl administration.

Discussion
Several animal models of SSc have been proposed in the literature.Among these models is the mouse intradermally injected with HOCl 9,11 .We performed an in-depth characterization of the proposed HOCl-SSc model, a model strongly favoured in the literature.Over the course of 6 weeks, skinfold, and dermal thickness of HOCl mice increased approximately 2-and threefold, respectively.Skin histology was characterized by patchy areas of fibrosis adjacent to unaffected skin.Consistent with these observations, the skin of HOCl-treated mice showed upregulated expression of fibrosis-related genes including Col1a1, Col3a1, and Acta2.Similarly, BLM-MP mice showed evidence of skin fibrosis.However, there was a striking lack of changes in the lungs; there were no differences in static compliance, histology, expression of pro-fibrotic genes, and immune cell compositions between PBS and HOCl-treated animals.This contrasted the findings of the BLM-MP model which showed lung inflammation and evidence of patchy fibrosis on histology.Micro-CT abnormalities and significant lung function impairment was also observed in BLM animals.Additionally, neither AOPPs nor anti-Scl70 antibodies were elevated in the sera of HOCl-treated mice.Furthermore, to test the hypothesis that mice susceptible to oxidative damage show enhanced lung fibrosis following HOCl administrations, experiments were conducted using the Nrf2 −/− mouse.Nrf2 −/− mice that received HOCl injections had comparable skin fibrosis, lung function, lung histology and BALF cellularity to Nrf2 −/− controls.
Several studies have reported that HOCl induces a gradual increase in skinfold and dermal thickness within a range similar to our results 12,35 .H&E-and MT-stained skin sections from HOCl animals showed a pattern of fibrotic injury characterized by spatial heterogeneity, epidermal hyperplasia and hyperkeratosis, consistent with prior reports 10,12,35 .These changes are also seen in SSc patients 36 .In some sections fibrosis was predominantly localized to the deep reticular dermis rather than to the papillary dermis 35 .It is important to note that the HOCl-induced skin manifestations were only observed in the lower back of the animal where injections were administered and not elsewhere, indicating a lack of a generalized fibrotic cutaneous response.The BLMinduced skin lesions also produced a phenotype resembling SSc, however, the observed effect was not restricted to the site of the minipump.
Following 6 weeks of HOCl injections, the lung function of BALB/c mice remained unchanged which corresponded with the lack of morphological alterations in the lung.Similarly, HOCl injections failed to generate lung fibrosis in C57BL/6 mice.Our findings are in contrast to several studies that report evidence of pulmonary fibrosis using similar methods 10,12,13 .We also performed automated and manual image analysis, which was not previously conducted, on PSR-stained lung sections and no differences were detected between PBS-and HOClinjected mice.Assessment of inflammatory cells in the BAL fluid did not show any evidence of an inflammatory response in HOCl-injected mice.Several technical factors such as inhomogeneous lung inflation resulting in areas of atelectasis and thick histological sections may suggest architectural abnormalities that could lead to erroneous interpretations of lung histology.Additional factors that could account for the discrepancies between our outcomes and published results include housing facility aspects (e.g.sterility, temperature, stressors, enrichments provided, etc.) and/or differences in mice purchased from different vendors.
Despite the lack of evidence for pulmonary injury, we characterized macrophage subsets in PBS and HOCltreated mice, given their role in bleomycin-induced lung injury and fibrosis 37,38 .We addressed the three subsets, tissue resident alveolar macrophages (TR-AM), monocyte-derived alveolar macrophages (Mo-AM), and interstitial macrophages (IM) 37 .Under normal conditions TR-AM are the most abundant immune cell in the lung.Conversely, in the setting of lung inflammation, circulating monocytes are recruited to the lung via the activation of chemokine receptor 2 (CCR2) and differentiate into Mo-AMs 37 .Initially Mo-Ams drive the inflammatory and fibrotic response; however, during repair it is proposed that Mo-Ams can either differentiate into cells that phenotypically resemble TR-AM or undergo apoptosis 37 .Several groups reported that depletion of circulating monocytes by intratracheal administration of liposomal clodronate 39 or using monocyte-chemoattractant protein-1 chemokine receptor knockout animals (CCR2 −/− ) 40 attenuated bleomycin-induced lung injury.These findings show that monocytes contribute to facilitating the progression of lung fibrosis.It has been suggested that monocytes differentiate into Mo-Ams and in combination with TR-AM acquire a profibrotic phenotype 37 .Indeed, inducing cell death of Mo-AM ameliorates the severity of lung fibrosis 38 .Following 42 days of HOCl injections, an increase in the number of Mo-AM was expected given the pathobiologic role of Mo-Ams in fibrosis 38 .During the fibrotic phase of bleomycin-induced lung injury, a decrease in the number of interstitial macrophages and an increase in the number of Mo-AM has been reported 37 .This is likely attributed to the large inflammatory response induced by bleomycin which causes the loss of most macrophages present under homeostatic conditions (TR-AM and IM) and their replacement by Mo-AM.The absence of an inflammatory response in our HOCl model contrasts with a previous report which showed increased CD4 + and CD8 + T cells, and CD19 + B cells in the lung by immunohistochemical analysis 17 .Moreover, we did not observe an upregulation of profibrotic genes in HOCl-injected mice whereas the mRNA expression of αSMA, TGF-β1, and collagen type I and III were upregulated in previous studies 4,12 .
HOCl has been proposed to generate lung fibrosis through excess formation of reactive oxygen species (ROS) and the induction of oxidative stress.Specifically, oxidized serum proteins (i.e.AOPPs) were suggested to be involved in the propagation of oxidative stress from the skin to the lungs 9 .However, our experiments failed to

Figure 3 .
Figure 3.At day 28 the BLM minipump model leads to impaired lung mechanics with interstitial inflammation and fibroblastic foci.(a) Mean pressure-volume (P-V) loops for PBS and BLM-treated mice.(b) Static lung compliance (C st ).(c) Lung resistance (Rrs) and elastance (Ers) (d) calculated by the flexiVent software by fitting the data from the single frequency forced oscillation manoeuvre to the single-compartment model.n = 5-10 mice per group, graphs show mean ± SEM.Two tailed t test, *p < 0.005 (e).Hematoxylin and eosin (HE) and Masson's Trichrome (MT)-stained lung sections.First row of HE and MT × 20 images and bottom row × 40 magnification.Scale bar: 50 μm.(f) Image analysis on PSR-stained slides and (g).Abnormal lung areas quantified by the Positive Pixel Count V9 Aperio Algorithm (airways and large vessels were excluded).Three transverse sections per left lung were analyzed (lower, middle, upper lobe).Mann-Whitney U Test was used for comparisons.ns not significant.

Figure 6 .
Figure 6.HOCl injections induce comparable skin fibrosis in Nrf2 −/− and WT control animals.(a) Skin thickness in mice increases over the course of 6 weeks of HOCl injections (n = 4-6).(b) Average measurements of dermal thickness (n = 4-6).Representative images of (c) H&E and (d) PSR staining of Nrf2 −/− and C57 HOCl-treated mice.H&E sections of both Nrf2 −/− and C57 mice show epidermal hyperplasia and hyperkeratosis indicated by the black arrow (H&E staining, panels i) and loss of adipose tissue/appendages in certain areas with preservation of them in adjacent areas (H&E staining, panels ii).Black arrows in the PSR-stained sections show representative dermal measurements.Data are represented as mean ± SEM from 2 experiments.Data were analyzed using Two-way ANOVA (a) or Mann-Whitney U test (b).ns not significant.
The lungs from PBS and HOCl mice were hydrolyzed in 10N NaOH for